Restriction Endonuclease Digestion of DNA from E. coli cells and Analysis by Agarose Gel Electrophoresis
The main goals of this experiment are testing an alternative procedure called “boiling lysis”, evaluating the quality of the purified plasmid for restriction digests, and identifying the mislabeled plasmid. The plasmid DNA from a carrier E. coli strain was purified by the boiling lysis. In the boiling lysis method, the bacterial cells were given momentary heat treatment in boiling water in presence of lysozyme and triton X-100. Since the plasmid DNA is small in size, it comes out from the bacterial cell, while the genomic DNA remains trapped in the cell. Successive high-speed centrifugation separated the plasmid DNA from rest of the cell debris, which form pellet. The pellet was removed and plasmid DNA was recovered by isopropanol precipitation method. Restriction enzymes are enzymes that selectively cut a DNA molecule at a particular place called restriction sites. Restriction enzymes also recognize a specific sequence of nucleotides, which vary between 4 and 8 nucleotides, in particular, palindromic. These restriction enzymes were kept in a 50% glycerol buffer that does not freeze at -20 oC. The four available enzymes used for this experiment were EcoRI, AvaI, HincII, and RsaI. The lengths of the DNA fragments from the restriction digests are used to map the relative locations of the four available enzymes in the plasmid. From this restriction sites, the unknown plasmid will be identified. Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. The length of the fragments from digestion is estimated by comparing the results obtained from the reference DNA fragments.
Materials and Methods
Plasmid purification. The bacterial culture used for this experiment is JM109 strain of E. coli carrying an unknown plasmid. For 20 seconds, 1.5 mL of bacterial culture was centrifuge at maximum speed in a microcentrifuge to pellet the bacteria. Before lysing the bacteria, 500 L of DNase-free water was used. The bacterial pellet was resuspend in 350 L of STET solution (8 % w/v sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 5 % w/v Triton X-100, 700 g/mL lysozyme, and 250 g/mL RNase A.). The 700 g/mL lysozyme from chicken egg was used to weaken the bacterial cell-wall peptidoglycan. Triton X-100 and heat disrupt the membrane lipid bilayer. The bacterium was fully lysed after it was placed in a boiling water for 1 minute. The proteins, such as DNase, RNase, lysozyme and all of the proteins from the lysed cell were extracted by adding 350 L of phenol:chloroform:isoamyl alcohol solution (24:24:1), vortexing for 5 seconds, and centrifuging for a minute in a microcentrifuge at maximum speed. The standard conditions for ethanol precipitation are 0.3 M KOAc with 2.5-3.0 volumes of absolute ethanol. The pellet was completely dried in a SpeedVac for 10 minutes, and then the pellet was dissolve in 20 L of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) for the final suspension.
DNA characterization. The plasmid DNA was diluted by adding 2 L of the plasmid DNA from plasmid purification with 500 L of TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). The wavelengths used in this experiment are 230, 260, and 280 nm. The spectrophotometer model used in the experiment is Beckman Coulter DU 730 Life Science UV/Vis Spectrophotometer. The equation used for calculating DNA concentration is as follows: [DNA] (g/mL) = A260 x 50.
Restriction digest methods. In each of the restriction digests, it contained 1 L of 10X NEB enzyme buffer (1X is 20 mM Tris-acetate, 50 mM potassium-acetate, 10 mM magnesium-acetate, 1 mM Dithiothreitol pH 7.9 at 25 oC), 1 L of BSA (10 mg/mL), 1 L of restriction enzyme (for double digests, 1 L of each enzyme was added), and 1 g of plasmid DNA. Subsequently all the components for the...
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